Catalytic and Structural Role of a Conserved Active Site Histidine in Berberine Bridge Enzyme

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• 作者：Silvia Wallner ; Andreas Winkler ; Sabrina Riedl ; Corinna Dully ; Stefanie Horvath ; Karl Gruber ; Peter Macheroux
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：August 7, 2012
• 年：2012
• 卷：51
• 期：31
• 页码：6139-6147
• 全文大小：427K
• 年卷期：v.51,no.31(August 7, 2012)
• ISSN：1520-4995

文摘

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1鈥揅2鈺怬 locus via interaction with the hydroxyl group at C2鈥?of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.